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Vehicle overload is detrimental to bridges and traffic safety. This paper presents a study on the fatigue performance of typical reinforced concrete (RC) beams of highway bridges under vehicle overload. A definition method of vehicle overload and a construction method of overload ladder spectrum were first proposed based on traffic data acquisition, statistical analysis and structural calculation of the highway bridges in Guangzhou. A fatigue experimental method was also proposed with the three-ladder vehicle overload spectrum, and the fatigue tests of 15 RC beams strengthened with carbon fiber reinforced polymer (CFRP) under three loading levels were then carried out. The fatigue performance and the failure mechanism of the strengthened beams were presented and discussed, and two fatigue life prediction methods were proposed with the established modified Palmgren-Miner rule and the loading level equivalent method respectively. The results showed that the fatigue performance of the strengthened RC beams was severely degraded under overload ladder spectrum compared with that under constant amplitude cyclic load, and the life prediction methods were proved effective. [ABSTRACT FROM AUTHOR]